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A Novel Truncating Mutation in HOMER2 Causes Nonsyndromic Progressive DFNA68 Hearing Loss in a Spanish Family

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A Novel Truncating Mutation in HOMER2 Causes Nonsyndromic Progressive DFNA68 Hearing Loss in a Spanish Family G C A T T A C G G C A T genes Article A Novel Truncating Mutation in HOMER2 Causes Nonsyndromic Progressive DFNA68 Hearing Loss in a Spanish Family María Lachgar 1,2,† , Matías Morín 1,2,† , Manuela Villamar 1,2, Ignacio del Castillo 1,2 and Miguel Ángel Moreno-Pelayo 1,2,* 1 Servicio de Genética, Hospital Universitario Ramón y Cajal, IRYCIS, Carretera de Colmenar km 9.100, 28034 Madrid, Spain; [email protected] (M.L.); [email protected] (M.M.); [email protected] (M.V.); [email protected] (I.d.C.) 2 Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 28034 Madrid, Spain * Correspondence: [email protected] † These authors have contributed equally to this work. Abstract: Nonsyndromic hereditary hearing loss is a common sensory defect in humans that is clinically and genetically highly heterogeneous. So far, 122 genes have been associated with this disorder and 50 of them have been linked to autosomal dominant (DFNA) forms like DFNA68, a rare subtype of hearing impairment caused by disruption of a stereociliary scaffolding protein (HOMER2) that is essential for normal hearing in humans and mice. In this study, we report a novel HOMER2 variant (c.832_836delCCTCA) identified in a Spanish family by using a custom NGS targeted gene panel (OTO-NGS-v2). This frameshift mutation produces a premature stop codon that may lead in the absence of NMD to a shorter variant (p.Pro278Alafs*10) that truncates HOMER2 at the CDC42 binding domain (CBD) of the coiled-coil structure, a region that is essential Citation: Lachgar, M.; Morín, M.; for protein multimerization and HOMER2-CDC42 interaction. c.832_836delCCTCA mutation is Villamar, M.; del Castillo, I.; placed close to the previously identified c.840_840dup mutation found in a Chinese family that Moreno-Pelayo, M.Á. A Novel truncates the protein (p.Met281Hisfs*9) at the CBD. Functional assessment of the Chinese mutant Truncating Mutation in HOMER2 Causes Nonsyndromic Progressive revealed decreased protein stability, reduced ability to multimerize, and altered distribution pattern DFNA68 Hearing Loss in a Spanish in transfected cells when compared with wild-type HOMER2. Interestingly, the Spanish and Chinese Family. Genes 2021, 12, 411. https:// frameshift mutations might exert a similar effect at the protein level, leading to truncated mutants doi.org/10.3390/genes12030411 with the same Ct aberrant protein tail, thus suggesting that they can share a common mechanism of pathogenesis. Indeed, age-matched patients in both families display quite similar hearing loss Academic Editors: Santiago phenotypes consisting of early-onset, moderate-to-profound progressive hearing loss. In summary, Rodriguez and Laura Crisponi we have identified the third variant in HOMER2, which is the first one identified in the Spanish population, thus contributing to expanding the mutational spectrum of this gene in other populations, Received: 20 December 2020 and also to clarifying the genotype–phenotype correlations of DFNA68 hearing loss. Accepted: 9 March 2021 Published: 12 March 2021 Keywords: hereditary hearing loss; next-generation sequencing; custom panel; HOMER2; CDC42 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. Hearing loss is the most common sensory deficit in humans, affecting around 1 in 1000 newborns. Its prevalence increases with the age up to 6–8% in the adult population, having a strong impact on the individual’s social isolation [1]. Genetic causes account for 50–60% of newborn hearing loss, 30% of which are nonsyndromic forms of deafness [2]. The genetic Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. etiology of hearing impairment is highly heterogeneous. Up to date, 160 nonsyndromic This article is an open access article sensorineural hearing loss (NSSNHL) loci have been mapped, of which 122 genes have been distributed under the terms and identified [3]. Sixty-seven of these loci and 50 of these genes are associated with autosomal conditions of the Creative Commons dominant NSSNHL, being, in most cases, rare forms of post-lingual and progressive Attribution (CC BY) license (https:// hereditary hearing impairment. creativecommons.org/licenses/by/ Autosomal dominant NSSNHL-linked genes encode proteins with a wide variety of 4.0/). functions [4], such as cytoskeleton proteins (ACTG1, DIAPH1, PLS1), adhesion proteins (GJB2, Genes 2021, 12, 411. https://doi.org/10.3390/genes12030411 https://www.mdpi.com/journal/genes Genes 2021, 12, 411 2 of 9 GJB3, GJB6, TJP2), motor proteins (myosins), scaffolding proteins (HOMER2), extracellular matrix proteins (TECTA, COL11A2), proteins involved in ion homeostasis, such as ionic channels (KCNQ4), transcription factors (EYA4, POU4F3), and even a microRNA (MIR96)[5]. The HOMER2 gene maps to chromosome 15q24.3 [6] within the DFNA68 critical interval and consists of nine exons. Two human transcript variants have been described (NM_004839.4 and NM_199330.3) as encoding the short isoform 1 (NP_004830.2, 343aa) and the long isoform 2 (NP_955362.1, 354aa) of HOMER2, respectively. HOMER2 belongs to a protein family encompassing three members: HOMER1 (MIM604798), HOMER2/CUPIDIN (MIM604799), and HOMER3 (MIM604800). Like HOMER2, members 1 and 3 of the family have long and short isoforms due to alternative splicing [7]. HOMER family members are scaffolding proteins that play a key role in Ca2+ signaling [8–10], mostly at the Post- Synaptic-Densities (PSD) [11], where they interact with G-protein coupled metabotropic glutamate receptors (mGluRs) [12] and regulate excitatory signal transduction and re- ceptor plasticity [7]. In their structure, HOMER proteins present a conserved N-terminal domain known as Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) homology 1 (EVH1) domain [7,13] that binds to proline-rich sequences (i.e. Pro-Pro-x-x-Phe, Pro-x-x- Phe or Leu-Pro-Ser-Ser-Pro, where x represents any amino acid) and a C-terminal domain that consists of a coiled-coil (CC) structure that includes a CDC42 binding domain (CBD) and two Leucine Zipper (LZA and LZB) motifs [7,14]. This C-terminal fragment mediates self-association with other Homer family members [7] and the interaction with the small GTPase CDC42 [15] through its CBD. In mice, Homer2 is widespread in the developing and maturing brain [16]. Recently, a study has shown that this gene is also expressed in a wide variety of developing tissues, including tooth, eye, cochlea, salivary glands, olfactory and respiratory mucosae, bone, and taste buds, being highly concentrated at puncta [17]. HOMER2 exhibits overlapping distribution patterns with HOMER1 and HOMER3, although they are distributed at distinct subcellular domains in several cell types [17,18]. Within the inner ear, HOMER2 is expressed in the stria vascularis, the Reissner’s membrane, and the inner and outer hair cells of the organ of Corti, especially in the stereocilia but also in perinuclear puncta and the cytoplasm [17,18]. Mild expression is observed in the vascular endothelium of the cochlea and the spiral ganglion [12]. Mice homozygous for the targeted deletion of Homer2 display early-onset rapidly progressive hearing loss [18]. HOMER2 was firstly associated with hearing loss by Azaiez et al. [18] who identified a heterozygous missense variant (p.Arg196Pro) in a European ascent family. A second mutation in HOMER2 (c.840_840dup; p.Met281Hisfs*9) was identified by Lu et al. [19] by Whole Exome Sequencing (WES) in a Chinese family segregating with hearing loss. Here, we present a third variant in HOMER2 (c.832_836delCCTCA, p.Pro278Alafs*10, NM_199330.3). This variant represents the second truncating mutation that affects the CBD and lead, as in the previous two families, to post-lingual and progressive hearing loss. This mutation is the first one identified in the Spanish population, thus increasing the mutational spectrum of this gene associated with DFNA68, a rare form of autosomal dominant nonsyndromic progressive hearing loss. 2. Patients and Methods 2.1. Patients Selection Patients and healthy relatives of family S1074 (Figure 1A) were recruited from the Uni- versity Hospital Ramón y Cajal (Madrid-Spain). Clinical history ruled out environmental factors as the cause of the hearing loss in the probands, and physical examination did not reveal any evidence of syndromic features. No other clinically significant manifestations, including balance or visual problems, were reported by any of the affected individuals. The hearing level was evaluated through pure tone audiometry. Air conduction thresholds were determined at frequencies ranging from 250 to 8000 Hz according to standard protocols. This study was designed in compliance with the tenets of the Helsinki Declaration, and patient enrolment was approved by the ethics committee and the human research Institu- Genes 2021, 12, 411 3 of 9 tional Review Boards of Hospital Ramón y Cajal (IRB number: 288-17). All participants of the family approved of the study and signed the Informed Consent. 2.2. Sample Collection A peripheral blood sample from each subject of the family S1074 enrolled in the study was collected by venipuncture in 5 mM EDTA tubes and genomic DNA was extracted using Chemagen MSM I (Magnetic Separation Module I, PerkinElmer, Massachusetts, MA, USA) according to the manufacturer’s instructions. DNA was quantified by the fluorometric method Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Massachusetts, MA, USA). 2.3. Targeted
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